BACKGROUND Improved denitrification with methane as alternative electron donor is regarded as a promising low-cost approach for organic nitrate-containing wastewater treatment. However, in situ capture of methane released from a methanogenic system for enhancing nitrogen removal seems to be scarcely reported. The applications of simultaneous denitrification, methanogenesis, and aerobic methane oxidation coupled to denitrification provide the possibility for solving the challenging issue. RESULTS In this study, simultaneous methanogenesis, aerobic methane oxidation, and denitrification (SMAMOD) was firstly established in a microaerobic up-flow sludge bed biofilm reactor (MUSBBR) treating high-strength synthetic wastewater with 2000 mg L-1 Chemical oxygen demand (COD) and 400 mg L-1 nitrate. The operation demonstrated 98.8% COD, 98.8% NO3--N, and 93.5% TN removals were stably obtained. Microbial community found the coexistence and cooperation of Methanothrix as methanogens, Methylogaea as aerobic methanotrophs, as well as facultative denitrification genera in SMAMOD system. Gene profiling demonstrated key functional genes involved in aerobic and anaerobic fermentation, methanogenesis and aerobic methane oxidation, as well as nitrate reduction induced by nitrate reductase systems. In organic oxygen-limited biotransformation, glucose was degraded mainly through aerobic respiration, anaerobic fermentation into methane by decarboxylation of acetic acid, and further methane aerobic oxidation pathways, while acetate- and methanol-denitrification and dissimilatory nitrate reduction to ammonium pathways participated in the nitrogen bioconversion. Further, the metabolic mechanism of SMAMOD might be unraveled by spatial interactions of key metagenomes in core microbial consortiums in the MUSBBR reactor. CONCLUSION The study offers valuable metagenomic insights into SMAMOD as a potentially compact process for sustainable treatment of high-strength organic nitrate-containing wastewater. (c) 2020 Society of Chemical Industry